The long-term goals of the proposed experiments are to understand the parameters governing the assembly of myofibrils in embryonic cardiac muscle cells and those governing the maintenance and attachment of myofibrils in cardiomyocytes of adult animals. The major emphasis of the experimental approaches is on analyzing myofibrils inside living cells via the microinjection of fluorescently labeled cytoskeletal proteins and the use of quantitative optical techniques. The first specific aim is to analyze myofibrillogenesis in living isolated embryonic cardiac myocytes using video and 3D-confocal microscopy to test the hypothesis that the future myofibril is laid down in a shortened version, the premyofibril, that lengthens by the incorporation of myofibrillar proteins. The second aim will investigate the role of non-muscle myosin IIB during myofibrillogenesis to test the hypothesis that this molecule is responsible for the alignment of the initial short sarcomeric units of the premyofibril which will become the longer sarcomeres of the mature myofibrils. The third aim is to test the hypothesis that zeugmatin, a protein found in Z- Bands, is responsible for the fusion of the Z-Bodies of the pre- and nascent myofibrils into Z-Bands of the mature myofibrils. The fourth aim is to determine how myofibrils maintain their integrity during protein turnover by analyzing with microscopic methods where actin enters the sarcomere of adult cardiomyocytes. The fifth specific aim is to analyze the functional attachment of the surface Z-Bands to the cell membrane using a novel method of growing cells on a deformable rubber surface. Various microinjected probes will be used to test the hypothesis that these Z-bands are coupled to the surface via a system of integrin-vinculin-alpha-actin molecules. The advanced optical methods proposed to study isolated cardiac muscle cells allow hypotheses about the formation and repair mechanisms of myofibrils to be tested directly in the living cardiac myocyte. An integrated microscopic approach will be used to follow individual cardiomyocytes from the light to the electron microscopic level. These approaches should yield new facts about basic processes in cardiac myocytes isolated from embryonic and adult hearts.